Ionotropic human glutamate receptor subunit NR3

Abstract

Neurotransmission by excitatory amino acids (EAAs) such as glutamate is mediated via membrane-bound surface receptors. This neurotransmission has been found to be modulated by certain modulatory proteins. DNA coding for a family of such modulatory proteins has now been isolated and the modulatory proteins have been characterized. Herein described are recombinant cell lines which produce these modulatory proteins as heterologous membrane-bound products. Also described are related aspects of the invention, which are of commercial significance, including the use of cell lines which express the modulatory proteins either homomerically, or hotoromorically in a complex with an NMDA receptor, as a tool for discovery of compounds which affect the function of the modulatory proteins.

We claim:  
     
       1. A method of assaying a candidate ligand for interaction with a human NR3 protein selected from the group consisting of: 
       NR3-1 having the amino acid sequence of SEQ ID NO:2; and  
       NR3-2 having the amino acid sequence of SEQ ID NO:2 with the exception that the serine residue at position 407 is an asparagine residue,  
       which comprises the steps of incubating the candidate ligand under appropriate conditions with a cell having incorporated expressibly therein a heterologous polynucleotide encoding said NR3 protein, or with a membrane preparation derived therefrom, and then determining the extent of binding between the human NR3 protein and the candidate ligand. 
     
     
       2. A method of assaying a candidate ligand for interaction with a human heteromeric receptor complex comprising human NR3 protein and human NMDA protein, wherein said NR3 protein is selected from the group consisting of: 
       NR3-1 having the amino acid sequence of SEQ ID NO:2; and  
       NR3-2 having the amino acid sequence of SEQ ID NO:2 with the exception that the serine residue at position 407 is an asparagine residue,  
       said method comprising the steps of incubating the candidate ligand under appropriate conditions with a cell that has been engineered genetically to produce said heteromeric receptor complex, said cell having incorporated expressible therein a heterologous polynucleotide encoding said NR3 protein and a heterologous polynucleotide encoding said NMDA protein, or with a membrane preparation derived therefrom, and then determining the extent of binding between the complex and the candidate ligand, or determining ligand-induced electrical current across said cell or membrane. 
     
     
       3. A method as defined in  claim 2 , wherein said human NMDA protein is selected from the group consisting of: 
       a) NMDAR1-1 having the amino acid sequence of SEQ ID NO:11;  
       b) NMDAR1-2 having the amino acid sequence of SEQ ID NO:11 wherein amino acids 841-850 of SEQ ID NO:11 are replaced by the amino acids encoded by SEQ ID NO:13;  
       c) NMDAR1-3A having the amino acid sequence of SEQ ID NO:11 wherein amino acids 841-850 of SEQ ID NO:11 are replaced by the amino acids encoded by SEQ ID NO:14;  
       d) NMDAR1-3C having the amino acid sequence of SEQ ID NO:11 wherein amino acids 841-850 of SEQ ID NO:11 are replaced by the amino acids encoded by SEQ ID NO:15;  
       e) NMDAR1-3B having the amino acid sequence of NMDAR1-3C wherein residue 470 is a lysine residue;  
       f) NMDAR1-4 having the amino acid sequence of SEQ ID NO:11 wherein the sequence from position 803 is replaced with the amino acid sequence of SEQ ID NO:17;  
       g) NMDAR1-5 having the amino acid sequence of NMDAR1-1 wherein the amino acids of 160-185 are replaced with the amino acid sequence of SEQ ID NO:18;  
       h) NMDAR1-6 having the amino acid sequence of NMDAR1-2 wherein the amino acids of 160-185 are replaced with the amino acid sequence of SEQ ID NO:18;  
       i) NMDAR1-7 having the amino acid sequence of NMDAR1-3 wherein the amino acids of 160-185 are replaced with the amino acid sequence of SEQ ID NO:18; and  
       j) NMDAR1-8 having the amino acid sequence of NMDAR1-4 wherein the amino acids of 160-185 are replaced with the amino acid sequence of SEQ ID NO:18.